The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial chloride channel that plays a key role in electrolyte and fluid homeostasis. Mutations in the CFTR gene cause cystic fibrosis, whereas overstimulation of the channel by bacterial enterotoxins in the gut leads to secretory diarrhea. Thus, understanding the mechanisms that regulate CFTR channel activity may be important in controlling these diseases. Evidence has accumulated that diverse binding interactions control both the maturation of CFTR and its transport to the plasma membrane. Different binding partners mediate different trafficking effects, and it is likely that the overall level of CFTR expression is determined by the interplay of their competing influences. Furthermore, many of these proteins contain multiple PDZ domains that bind to the C-terminus of the CFTR protein, and it appears that they can potentiate channel activity by inducing or stabilizing the formation of CFTR dimers. We propose a two-pronged approach to investigate the structural basis of these regulatory protein-protein interactions. First, we will investigate the binding parameters of a series of CFTR-binding PDZ domains that will be recombinantly expressed. Binding kinetics and thermodynamics will be determined using surface plasmon resonance and fluorescence polarization techniques, and the ability of the various domains to compete for CFTR binding sites assessed. For divalent constructs binding analysis will also include their ability to mediate CFTR dimerization both alone and in competition with other PDZ domains. Secondly, the stereochemistry of the binding interactions will also be determined crystallographically. Following structural comparisons, hypotheses as to the basis of differences in their binding characteristics will be tested by site-directed mutagenesis, by sequence variations in the target peptide, and ultimately by the identification of small-molecule modulators. These variations will then be tested both in vitro and in vivo. Ultimately, these structural insights may provide the basis for designing compounds that can influence disease progression by targeted stabilization or disruption of particular protein-protein interactions that regulate CFTR function.